ATP-gated P2X receptors (P2XRs) are a superfamily of ligand-gated ion channels (LGICs)1-3 that have recently become a focus of investigation in alcohol studies. This is due, in part, to the building evidence suggesting that P2XRs may be important mediators of ethanol-induced behavioral effects4-7.
P2XRs are broadly distributed in the central and peripheral nervous systems2,3,8. Currently, seven subunits of the P2X family of LGICs have been identified (P2X1-P2X7). P2XR subunits form functional ATP-activated homomeric channels (e.g., P2X2, P2X4) as well as heteromeric receptors (e.g., P2X2/3, P2X4/6) in mammals3,9. P2XR channels are multimeric proteins where a functional receptor results from the assembly of three subunits9-11. P2X subunits consist of two transmembrane (TM) domains, a large extracellular domain (ectodomain) and intracellular amino-(N) and carboxy (C)-terminals (for review see3,12). Recent crystallographic investigations confirmed the trimeric structure of P2XRs with TM2 domains lining the pore13. The TM1 and TM2 membrane spanning segments are involved in ion channel gating and ion pore formation14,15. The ectodomain contains an ATP-binding site and is a site for channel regulation2,3,16.
P2X2, P2X3 and P2X4Rs expressed in Xenopus oocytes are sensitive to ethanol at intoxicating concentrations17-19. Studies using a chimeric strategy that exploited the opposite effects of ethanol on P2X2R (ethanol inhibition) and P2X3R (ethanol potentiation) found that residues contained within the ectodomain-TM domain interfaces are important for mediating or modulating the threshold and magnitude of response of ethanol in P2X3Rs5. Extending the investigation to P2X4Rs resulted in the identification of two key residues at the ectodomain-TM2 interface (Asp331 and Met336) that, when substituted to alanine caused a significant reduction in ethanol (10-200 mM) inhibition of ATP-gated currents without causing marked changes in ATP Imax, EC50 or Hill slope6. Additional studies exploring the relationship between structure and ethanol action found a significant correlation between hydropathy, polarity and the % inhibition by 50 mM ethanol of the residues at position 3366. Preliminary investigations using an alanine mutagenesis strategy identified position 46 (Trp46) contained within the ectodomain-TM1 interface as a potential target for the action of ethanol. Taken together, the findings support the hypothesis that the ectodomain-TM interfaces contain residues that are important for the action of ethanol in P2X4Rs.
Ivermectin (IVM) is a well tolerated, broad spectrum anti-parasitic medication used in veterinary and human medicine20,21. Building evidence indicates that IVM can be used as a pharmacological tool for identifying the contribution of P2X4Rs in ATP-mediated processes22. Recent reports suggest that IVM binds at the lipid-protein interface acting on sites located in the TM segments at the ectodomain-TM domain interface of the P2X4R23,24. Individual mutations of residues at these interfaces indicated that positions Trp50, Val60 and Val357 may play an important role in IVM regulation of the rate of channel function23,24.
Alcohol abuse and alcoholism affect an estimated 17 million people, cause over 100,000 deaths, and cost nearly $200 billion annually in the United States36,37. To address these issues, considerable attention has begun to focus on the development of medications to prevent and treat ethanol abuse and dependence38-40.